DESCRIPTION: This proposal for competitive renewal of a long standing and productive project requests 5 years support for the analysis of unusual ankyrin and beta spectrin transcripts identified in muscle by the analysis of cDNA and genomic clones. Ankyrin and spectrin are two critical components of the cytoskeletons of many cells; their function and importance has been best characterized in erythroid cells. Like other components of the cytoskeleton, notably protein 4.1, ankyrin and spectrin have been found to consist of complex families of proteins arising from multiple genes that generate diverse proteins by means of selective transcription and alternative mRNA splicing. The functions and pathophysiologic importance of these cytoskeletal components in nonerythroid tissues are much less clear. Many of the unique features of the erythrocyte, such as its prolonged survival in the circulation without capacity for replacement of damaged proteins by new protein synthesis, complicate the applicability of erythrocytes as a model for other tissues. Dr. Barker and her associates plan to use normal mice and mice with mutations affecting akyrin and beta spectrin to study the expression, intracellular localization, function, and pathophysiology of these proteins in mouse muscle and brain. There are 5 Specific Aims. The first Specific Aim will utilize standard cloning technologies in an effort to establish detailed structural information about novel transcripts that this group has identified in muscle and brain. The second Specific Aim uses analogous methods of protein analysis in order to characterize the proteins generated from these mRNA's. The third Specific Aim will utilize immunocytochemistry, in situ into hybridization, and electron microscopic techniques to establish intracellular localization of these transcripts. The fourth Specific Aim will utilize a variety of techniques in attempts to identify the protein binding partners of these novel proteins. The primary methods will be immunoprecipatation and affinity purification. The final Specific Aim will attempt to use mice with documented deficiencies in these proteins to determine abnormalities of muscle and brain structure by light and electron microscopic methods, using antibodies generated in the earlier portions of the work plan.